Lettuce infectious yellows virus (LIYV) was first identified by Duffus et al., 1982, when it occurred in epidemic proportions in the desert Southwest of the United States. Large losses occurred in lettuce, sugarbeets, cantaloupe, watermelon, other melons, and squash due to stunting of infected plants and leaf rolling, yellowing, and brittleness. It has also been reported that LIYV infects cucumbers in Europe.
LIYV virus particles are flexuous and filamentous measuring approximately 1800-2000 nm in length. The LIYV genome is single-stranded RNA and includes about 15,700 nucleotides in positive (+, coding, or sense) polarity. Preliminary characterization of the RNA genome reveals that there may be two RNAs present in LIYV-infected plants and purified LIYV virions (Klaassen et al., 1992).
It has been shown for several viruses [tobacco mosaic virus (Powell-Abel et al., 1986), alfalfa mosaic virus (Tumer et al., 1987), cucumber mosaic virus (Cuozzo et al., 1988; Quemada et al., 1991), and potato virus X (Hemenway et al., 1988)] that expression of the coat protein gene in transgenic plants results in a plant which is resistant to infection by the respective virus. It has also been shown for tobacco vein mottle virus (TVMV) that expression of the protease gene (NIa) confers resistance to TVMV (Maiti et al., 1993). It has not been determined, however, whether this method for engineering resistance to infection will extend to different kinds of viruses such as LIYV. In addition, it has not been determined whether the expression of other viral proteins, such as the heat shock protein-70, in transgenic plants results in a plant which is resistant to infection by the respective virus.
The LIYV genes isolated from viral RNA do not contain the regulatory sequences needed for gene expression. For example, the LIYV coat protein gene does not contain the signals necessary for its expression once transferred and integrated into a plant genome. Therefore, the coat protein gene, like the RNA polymerase gene, the heat shock protein-70 gene, ORF 3 of LIYV RNA1 and ORF 6 of LIYV RNA2, must be engineered to contain a plant expressible promoter 5' to a translation initiation codon (ATG) and a plant functional poly(A) addition signal (AATAAA) 3' of a translation termination signal.
In a first embodiment of the present invention, the nucleotide sequence of the coat protein gene for LIYV, along with its putative amino acid sequence, has been determined. The gene has been inserted into expression vectors to supply it with the necessary genetic regulatory sequences so that the genes can be expressed when incorporated into a plant genome. Plant cells are transformed with the vector construct and the plant cells are induced to regenerate sexually mature plants. The resulting plants contain the coat protein gene and possess an increased resistance to infection by the virus from which the coat protein gene is derived.
In a second embodiment of the present invention, the nucleotide sequence of the heat shock protein-70 gene for LIYV, along with its putative amino acid sequence, has been determined. The gene has been inserted into expression vectors to supply it with the necessary genetic regulatory sequences so that the genes can be expressed when incorporated into a plant genome. Plant cells are transformed with the vector construct and the plant cells are induced to regenerate sexually mature plants. The resulting plants contain the heat shock protein-70 gene and possess an increased resistance to infection by the virus from which the heat shock protein-70 gene is derived.
In a third embodiment of the present invention, the nucleotide sequence of the RNA polymerase gene for LIYV, along with its putative amino acid sequence, has been determined. The gene has been inserted into expression vectors to supply it with the necessary genetic regulatory sequences so that the genes can be expressed when incorporated into a plant genome. Plant cells are transformed with the vector construct and the plant cells are induced to regenerate sexually mature plants. The resulting plants contain the RNA polymerase gene and possess an increased resistance to infection by the virus from which the RNA polymerase gene is derived.
In a fourth embodiment of the present invention, the nucleotide sequence of ORF 6 of LIYV RNA2, along with its putative amino acid sequence, has been determined. The gene has been inserted into expression vectors to supply it with the necessary genetic regulatory sequences so that the genes can be expressed when incorporated into a plant genome. Plant cells are transformed with the vector construct and the plant cells are induced to regenerate sexually mature plants. The resulting plants contain the LIYV RNA2 ORF 6 gene and possess an increased resistance to infection by the virus from which the LIYV RNA2 ORF 6 gene is derived.
In a fifth embodiment of the present invention, the nucleotide sequence of ORF 3 of LIYV RNA1, along with its putative amino acid sequence, has been determined. The gene has been inserted into expression vectors to supply it with the necessary genetic regulatory sequences so that the genes can be expressed when incorporated into a plant genome. Plant cells are transformed with the vector construct and the plant cells are induced to regenerate sexually mature plants. The resulting plants contain the LIYV RNA1 ORF 3 gene and possess an increased resistance to infection by the virus from which the LIYV RNA1 ORF 3 gene is derived.